Liquid chromatography typically refers to separation processes in which a solution of dissolved substances is passed through a tube, called a chromatographic column, filled with a porous granules, called a stationary phase. As the liquid leaves the tube, the substances are separated into single components due to their attraction to the porous medium in the tube. In other words, as a dissolved mixture passes through a chromatographic column, the molecules of substances being separated reside for some time in the stationary phase (where their flow rate essentially stops) and for some time in the mobile liquid phase (where they flow at the same rate as the liquid phase).
Among the variety of chromatographic techniques, there are two general types of liquid chromatography: adsorption chromatography and size exclusion chromatography. In adsorption chromatography the components are separated due to chemical or physical interaction between the components to be separated and the stationary phase. The amount of time that molecules spend in either the liquid or solid phases within the chromatographic column depends on their chemical or physical attraction to the solid phase particles. The molecules that are more readily attracted reside in the stationary phase longer than those that are less easily attracted; as a result, they move through the chromatographic column more slowly, and thus the separation of mixtures into individual components can be effected.
In size exclusion chromatography, the separation of components is a function of their molecular size and the stationary phase typically does not attract the components. Separation depends on the amount of time that the substances spend in the porous stationary phase as compared to time in the fluid. The probability that a molecule will reside in a pore depends on the size of the molecule and the pore. In addition, the ability of a substance to permeate into pores is determined by the diffusion mobility of macromolecules which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all; and, for very small macromolecules the probability of penetration is close to unity. While components of larger molecular size move more quickly past the stationary phase, components of small molecular size have a longer path length through the pores of the stationary phase and are thus retained longer in the stationary phase.
Size exclusion chromatography is widely utilized in a variety of scientific fields. In the biological sciences, size exclusion chromatography is used for the isolation and purification of biological molecules, such as peptides, hormones or DNA. Size exclusion chromatography is used in the polymer chemistry field to determine molecular weight distribution of polymers and to isolate or resolve polymers of a particular size from a mixture of variously sized polymers.
A variety of stationary phases have been developed for use in size exclusion chromatography, dextran, cross-linked polymers of styrene-divinylbenzene, acrylamide or vinylacetate, or macroporous inorganic material, such as silica, activated charcoal, or alumina. Although such stationary phases are widely used, a number of drawbacks limit their usefulness.
Although polymer-based materials are generally used as the stationary phase in size exclusion chromatography, their usefulness is limited. For instance, high flow rates desirable with modern high pressure chromatographic systems are generally not possible with polymeric stationary phases. And, the choice of solvents that can be used as the mobile phase in polymer-based stationary phase size exclusion chromatography columns is limited by the solubility of a polymeric stationary phase. Many otherwise useful solvents cannot be used because they either degrade polymeric stationary phases or shrink or swell the polymer thereby disrupting the separation characteristics and capabilities of the sorbent. Even standard solvents such as water or methanol cannot be used with some polymeric stationary phases.
Inorganic materials, e.g. silica, charcoal and alumina, have also been employed as the stationary phase for size exclusion chromatography without some of the problems associated with polymeric materials. But inorganic sorbents present disadvantages of their own. For instance, silica particles are not suitable for use with alkaline solvents. The reactivity of silica particles also provides the opportunity for undesirable interaction between the chemical mixture and the silica particles.
Alumina particles have not been widely employed as a stationary phase for size exclusion chromatography because generally available alumina particles have too narrow a pore size distribution to effectively separate a wide range of macromolecules. Moreover, alumina particles that have been used for chromatography have not allowed sufficiently good flow rate, resulting in restricted flow of the mobile phase through the stationary phase.
U.S. Pat. Nos. 4,822,593; 4,900,537 and 5,037,795 disclose alumina particles comprising crystals extending radially outward from a central core, e.g. gaving wedge-shaped macropores, and their use for certain chromatographic applications. Chromatographic columns packed with such particles are available from Biotage, Inc. (Charlottesville, Va.); in columns for normal phase chromatography the particles are uncoated; and in columns for reverse phase chromatography the particles are coated, e.g. with polybutadiene. The use of such particles for separating macromolecules according to size has not been suggested nor is any discussion of the usefulness of such particles for size exclusion chromatography found in the literature.
Modern chromatographic techniques use high pressure to decrease the time necessary to analyze a chemical composition. When high pressure is used it is important that the flow of the mobile phase through the stationary phase be as unrestricted as possible. Some materials currently used for size-exclusion chromatography allow the development of significant "back pressure" in the column which restricts the flow of liquid through the column, decreasing the efficiency of the separation. The creation of a back pressure is an even more significant problem for size exclusion chromatography when attempted on a large scale, e.g. as a separation process.
There is, therefore, a need in the chromatographic industry for a stationary phase material for use in size exclusion chromatography that can more quickly and effectively separate macromolecules of a wide range of molecular weights with a variety of solvents without degrading the stationary phase or reducing its separation capabilities. There is also a need for a size exclusion chromatographic stationary phase that exhibits improved flow rate capabilities.